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Molecules
2024 Nov 04;2921:. doi: 10.3390/molecules29215214.
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The Inhibitory Effect and Mechanism of the Histidine-Rich Peptide rAj-HRP from Apostichopus japonicus on Human Colon Cancer HCT116 Cells.
Zhang Y
,
Gao S
,
Mao J
,
Song Y
,
Wang X
,
Jiang J
,
Lv L
,
Zhou Z
,
Wang J
.
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Colon cancer is a common and lethal malignancy, ranking second in global cancer-related mortality, highlighting the urgent need for novel targeted therapies. The sea cucumber (Apostichopus japonicus) is a marine organism known for its medicinal properties. After conducting a bioinformatics analysis of the cDNA library of Apostichopus japonicus, we found and cloned a cDNA sequence encoding histidine-rich peptides, and the recombinant peptide was named rAj-HRP. Human histidine-rich peptides are known for their anti-cancer properties, raising questions as to whether rAj-HRP might exhibit similar effects. To investigate whether rAj-HRP can inhibit colon cancer, we used human colon cancer HCT116 cells as a model and studied the tumor suppressive activity in vitro and in vivo. The results showed that rAj-HRP inhibited HCT116 cell proliferation, migration, and adhesion to extracellular matrix (ECM) proteins in vitro. It also disrupted the cytoskeleton and induced apoptosis in these cells. In vivo, rAj-HRP significantly inhibited the growth of HCT116 tumors in BALB/c mice, reducing tumor volume and weight without affecting the body weight of the tumor-bearing mice. Western blot analysis showed that rAj-HRP inhibited HCT116 cell proliferation and induced apoptosis by upregulating BAX and promoting PARP zymogen degradation. Additionally, rAj-HRP inhibited HCT116 cell adhesion and migration by reducing MMP2 levels. Further research showed that rAj-HRP downregulated EGFR expression in HCT116 cells and inhibited key downstream molecules, including AKT, P-AKT, PLCγ, P38 MAPK, and c-Jun. In conclusion, rAj-HRP exhibits significant inhibitory effects on HCT116 cells in both in vitro and in vivo, primarily through the EGFR and apoptosis pathways. These findings suggest that rAj-HRP has the potential as a novel targeted therapy for colon cancer.
Figure 1. Sequence, purification, and BLAST results of rAj-HRP. (a) The cDNA sequence and deduced amino acid sequence of rAj-HRP; (b) Tricine-SDS-PAGE analysis showing the purification of recombinant rAj-HRP. Lane 1: marker; Lane 2: uninduced expression of recombinant B21; Lane 3: induced expression of recombinant B21; Lane 4: purified rAj-HRP; (c) phylogenetic tree analysis of histidine-rich glycoprotein-like from rAj-HRP BLAST results. The text marked in red represents the histidine-rich peptide rAj-HRP from Apostichopus japonicus. The amino acid sequence of rAj-HRP shares around 60% similarity with histidine-rich glycoprotein-like from various species such as Pararge aegeria, Bicyclus anynana, Maniola jurtina, Maniola hyperantus, Plasmodium lophurae, Kryptolebias marmoratus, Stegodyphus dumicola, and Spodoptera litura.
Figure 2. Inhibitory effect of rAj-HRP on the proliferation and morphology of HCT116 cells. (a) CCK-8 assay results (n = 3) showing the inhibitory effect of rAj-HRP on HCT116 cell proliferation. Significant differences between the rAj-HRP-treated group and the control group (treated with empty medium) are indicated by *, ** p < 0.01, and *** p < 0.001. (b) Wright–Giemsa staining illustrating the effect of rAj-HRP on HCT116 cell morphology (Olympus digital imaging microscope, Tokyo, Janpan); 200× magnification; the bar indicates 2 μm.
Figure 3. Inhibition of HCT116 cell migration by rAj-HRP via ECM adhesion, cytoskeleton disruption, and MMP2 downregulation. (a) Effect of rAj-HRP on HCT116 cell migration towards bFGF: HCT116 cell migration was evaluated using the Transwell assay, with each experiment conducted in triplicate. (b) Significant differences in migration between groups treated with bFGF or rAj-HRP and the bFGF+ control group are marked by * (* p < 0.05, and ** p < 0.01; the bar indicates 2 μm. (c) The effect of rAj-HRP on the adhesion to ECM proteins: HCT116 cell adhesion to ECM proteins in the presence of rAj-HRP was quantified using the CCK-8 assay. Adhesion rates were calculated using the formula provided in the Materials and Methods Section. Each experiment was performed in triplicate. Significant differences in adhesion rates between the blank medium or rAj-HRP-treated groups are denoted by * (** p < 0.01, and *** p < 0.001). (d) Western blot results; the downregulation of MMP2 expression by rAj-HRP: Western blot analysis was conducted to determine MMP2 protein levels in HCT116 cells treated with either blank medium or rAj-HRP (4.13 μM, 5.37 μM, or 6.98 μM), with GAPDH as a control. (e) Statistical chart displaying the relative gray value of MMP2 (MMP2/GAPDH) (n = 3). Significant differences between the rAj-HRP-treated and control groups are represented by * (** p < 0.01, and *** p < 0.001). (f) Cells were or were not treated with rAj-HRP, and the cytoskeleton was stained using phalloidin–FITC. Significant differences between the rAj-HRP-treated groups and control groups are represented by * (** p < 0.01). (g) Disruption of the HCT116 cell cytoskeleton by rAj-HRP: HCT116 cell morphology was observed using differential interference contrast (DIC) microscopy (first column). The blue signal indicates nuclei stained with Hoechst 33258 (second column), while the green signal indicates F-actin stained with FITC–phalloidin (third column). The fourth column displays merged images of the second and third columns. Images were captured using a Zeiss laser scanning confocal microscope (ZEISS, Oberkochen, German) at 630× magnification. The bar indicates 10 μm.
Figure 4. rAj-HRP induces apoptosis in HCT116 cells by modulating PARP and BAX expression. (a) The effect of rAj-HRP on HCT116 cell apoptosis assessed by the TUNEL assay (Zeiss laser scanning confocal microscope, 630× magnification; the bar indicates 10 μm). (b) The effect of rAj-HRP on HCT116 cell apoptosis assessed by Hoechst staining (Zeiss laser scanning confocal microscope, 630× magnification; the bar indicates 10 μm). (c) The effect of rAj-HRP on PARP expression in HCT116 cells: (1) Western blot results; (2) statistical chart showing the relative gray value of PARP (PARP/GAPDH) (n = 3; significance between rAj-HRP-treated groups and control groups is indicated by *, * p < 0.05, and *** p < 0.001). (d) The effect of rAj-HRP on BAX expression in HCT116 cells: (1) Western blot results; (2) statistical chart showing the relative gray value of BAX (BAX/GAPDH) (n = 3; significance between rAj-HRP-treated groups and control groups is indicated by *, * p < 0.05, and ** p < 0.01).
Figure 5. The effect of rAj-HRP on tumor growth in HCT116 xenograft BALB/c mice. (a) The effect of rAj-HRP on body weight: changes in body weight of HCT116 xenograft mice treated with rAj-HRP or 5-Fu (n = 5). (b) The effect of rAj-HRP on tumor volume growth: tumor volume growth trends in HCT116 xenograft mice treated with rAj-HRP or 5-Fu (n = 5). (c) Tumor images: representative images of tumors from different treatment groups. Unfortunately, two of the five nude mice injected with 5-Fu died 10 days after being administered the drug. (d) Tumor volume effect: tumor volumes in mice treated with rAj-HRP or 5-Fu compared to the control group (n = 5). Significant differences are indicated by *, * p < 0.05; ** p < 0.01, and *** p < 0.001. (e) The effect on tumor weight: tumor weights in mice treated with rAj-HRP or 5-Fu compared to the control group (n = 5). Significant differences are indicated by *, * p < 0.05 and *** p < 0.001. (f) HE staining of tumor sections: hematoxylin and eosin (HE) staining of HCT116 xenograft tumor sections from mice treated with rAj-HRP, observed using a Nikon inverted fluorescence microscope (Nikon, Tokyo, Iapan) at 200× magnification; the bar indicates 2 μm.
Figure 6. The effect of rAj-HRP on EGFR and its downstream signaling pathways in HCT116 cells. (a) EGFR expression: Western blot analysis of EGFR in HCT116 cells using GAPDH as a loading control. The histogram displays the relative gray value of EGFR (EGFR/GAPDH). (n = 3; significant differences between rAj-HRP-treated groups and the control group are indicated by *, * p < 0.05, and *** p < 0.001). (b) P38 MAPK expression: Western blot results for P38 MAPK. The histogram displays the relative gray values of P38 MAPK (P38 MAPK/GAPDH). (n = 3; significant differences between rAj-HRP-treated groups and the control group are indicated by *, * p < 0.05, ** p < 0.01, and *** p < 0.001). (c) c-Jun expression: Western blot results for c-Jun. The histogram displays the relative gray values of c-Jun (c-Jun/GAPDH). (n = 3; significant differences between rAj-HRP-treated groups and the control group are indicated by *, ** p < 0.01, and *** p < 0.001). (d) AKT expression: Western blot results for AKT. The histogram shows the relative gray values of AKT (AKT/GAPDH). (n = 3; significant differences between rAj-HRP-treated groups and the control group are indicated by *, * p < 0.05, and ** p < 0.01,). (e) p-AKT expression: Western blot results for AKT. The histogram shows the relative gray values of p-AKT (p-AKT/GAPDH). (n = 3; significant differences between rAj-HRP-treated groups and the control group are indicated by *, * p < 0.05, and *** p < 0.001). (f) p-PLCγ expression: Western blot results for p-PLCγ. The histogram displays the relative gray value of p-PLCγ (p-PLCγ/GAPDH). (n = 3; significant differences between rAj-HRP-treated groups and the control group are indicated by *, ** p < 0.01, and *** p < 0.001).
